BACKGROUD
Gut microbiota imbalance is gaining interest for colorectal cancer (CRC) pathogenesis. In particular, bacterial toxins are emerging as key players in CRC development and are often found associated with tumor tissue. We recently showed that Cytotoxic Necrotizing Factor 1 (CNF1) from pathogenic Escherichia coli promotes colorectal carcinogenesis in a mouse model of inflammatory bowel disease. Accumulating evidence reveals that gut bacteria can regulate host epigenetic modifications, such as DNA methylation, through alterations in chromatin structure and function. Progressive epigenetic changes in normal colorectal epithelium contribute to CRC initiation and progression. Accordingly, microbiota-derived metabolites and toxins like CNF1 may serve as indirect epigenetic regulators, forging a critical link between gut microbiota, the host genome and tumorigenesis.
OBJECTIVE
Based on these premises, in the present study, we evaluated the methylation changes induced by CNF1 that may be associated to carcinogenesis.
METHODS
We profiled the DNA methylome of colon samples from mice given intrarectal CNF1, either alone or in combination with 2% with dextran sulfate sodium (DSS) over 6 months, using the Infinium Mouse Methylation BeadChip (Illumina Inc., San Diego, CA, USA). Differentially methylated probes (DMPs) and regions (DMRs) were identified for two comparisons -CNF1 versus untreated and DSS versus CNF1+DSS- via DMRcate R package. To explore the biological relevance of these methylation changes, Genes linked to DMPs/DMRs underwent Reactome pathway and Gene Ontology analyses using Enrichr.  Subsequently, we validated the results on CNF1-exposed CRC patients. Bisulfite pyrosequencing analyses and RNA sequencing are on-going for the validation of methylome results.
RESULTS
We found that intrarectal exposure to CNF1 induces DNA methylation changes, which are markedly enhanced in the presence of chronic inflammation. These alterations primarily target pathways regulating immune function, cellular growth, and inflammation, shifting toward cancer-related pathways in the presence of colitis. Furthermore, some genes showing differential methylation in CNF1-treated mice also exhibit changes in CRC patients harboring CNF1-producing bacteria.
CONCLUSIONS
Understanding CNF1's influence on host methylome further illuminates the role of the gut microbiota in CRC pathogenesis via non-mutational mechanisms and highlights potential therapeutic targets. Further studies should determine if CNF1 also induces other epigenetic modifications beyond DNA methylation.